Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 315-327
ISSN: 2319-7706 Volume 2 Number 5 (2013) pp. 315-327
http://www.ijcmas.com
Original Research Article
Studies on epidermal appendages of chick embryos
Hassan I.H. El-Sayyad1*, Yosra A. Fouda1, Soad A.Khalifa1,
Asma Suliman AL-Gebaly2 and Omnia K. R. El-Sayyad1
1
2
Department of Zoology, Faculty of Science, Mansoura University, Egypt
Department of Biology, Science College, Princess Noura Bint Abdul Rahman University,
Riyadh-Kingdom of Saudia Arabia
*Corresponding author e-mail: [email protected]
ABSTRACT
Keywords
Chick embryos;
skin;
Foot scales;
beak;
Amino acids;
Keratin;
light;
Scanning and
transmission
Electron
microscopy.
The present study investigated the chicken skin appendages (abdominal feathers,
beak and foot scales) at 9, 10, 12, 14 and 16 days old and revealed that keratin
content is detected early in foot scales at 10-days embryo and then markedly
detected at highest concentration in beak in developing embryos. The amino acid
contents, especially serine, glutamine, prolline , glycine, alanine, valine and leucine
are detected at higher concentration in the different epidermal appendages.
However the amino acids serine, glycine, alanine, leucine and isoleucine are more
increased in foot scales more than the other body regions. SDS-PAGE of the
studied regions exhibited marked keratin expression in foot scales more than dorsal
body skin. Scanning electron microscopy (SEM) exhibited similar developmental
origin. The feather filaments possess consequences of reconstruction with the
formation of main axon and barb. The barbs characterized by its articulated
cylindrical structural pattern. Dorsal body skin attained considerable thinning
comparing with foot scales as well as lack of cornification. Different types of scales
(reticulate, scutate and scutella) are detected during embryo development. TEM
observation exhibited increased accumulation of keratohyaline granules of different
forms in the cytoplasm of stratum granulosum of foot scales comparing with fine
collection of keratin filaments in dorsal body skin.
Introduction
Cancer is the abnormal growth of cells in
There are a variety of skin appendages,
including feathers, scales, claws, and
beaks in birds. Their morphogenesis
involves the transformation of the
primarily flat epidermis into specialized
complex structures for induction, cell fate
specification, proliferation, differentiation,
epithelial
cycling,
and intimate
interactions with the mesenchyme
(Chuong, 1998). Chicken skin offers
distinct patterns of different cutaneous
appendages,
developed
through
consequences of epithelial-mesenchymal
interactions (Choung and Widelitz, 1998;
Jiang et al., 1998). Variations in
developmental processes are thought to be
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Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 315-327
a key mechanism of organ novelty
(Chuong, 1998).
white leghorn hens were obtained from a
commercial supplier. The eggs were
incubated in a humidified incubator at 37
± 0.50C, 60% relative humidity being
turned three times daily. The chick
embryos were collected at 9,10, 12, 14 and
16 days old according to Hamburger and
Hamilton (1951).
The feathers of birds develop from
embryonic epidermal lineages that
differentiate during outgrowth of the
feather germ. Scutate scales, which consist
of peridermal layers, a subperiderm, and
an alpha stratum. The feather-type B
keratins are expressed in the subperiderm
cells of embryonic scutate scales, as well
as the barb ridge lineages of the feather.
However, unlike the subperiderm of
scales, which is lost at hatching, the cells
of barb ridges, in conjunction with
adjacent cell populations, give rise to the
structural elements of the feather (Sawyer
et al., 2003a). From the dermomyotome,
neural crest and somatopleura cells give
rise to form the dermis. They interact with
epithelium to form the skin and skin
appendages. During these processes,
regional specificities are endowed in
development and evolution to generate
diverse integuments and their appendages
(Wu et al., 2004). Jiang et al., (2004)
outlined several understand concerning
how do these patterns form. Are they
under strict genetic control? Then, why are
many patterns similar but not identical?.
Are they under epigenetic control? Then
why do patterns appear to be amazingly
consistent in animals of the same species?.
The present study outlined the variations
of developmental consequences of beak
structure, feathers, foot scales using
assaying of their contents of keratin and
amino acids and protein electrophoresis.
Light, scanning and transmission electron
microscopy studies were carried out to
clarify the diversity of their structural
pattern.
Keratin content
Fresh specimens of skin were separated
from the dorsum skin, foot and beak
region and kept in nitrogen solution at 80C. Keratin extractions were carried out
at the selected 9,12 & 14-days old embryo
according to Dreher et al., (1998). An
aliquot of 1 ml of 1 M NaOH was added to
a 2 ml cryo vial with the rolled tape strip,
and the tube was stored for at 4°C
overnight. The next morning, 1 ml of 1 M
HCl was added and the sample and mixed
thoroughly. A 100 µl aliquot of skin
sample was aliquoted with 100 µl of 1 M
NaCl and one milliliter of Bradford 's
reagent and allowed to stand at room
temperature for 2 min before measuring
absorbance at 595 nm using a UV
spectrophotometer (Beckman DU640;
Beckman Instruments, Palo Alto, CA). A
standard
curve
prepared
to
use
commercially available human keratin
(Sigma, St Louis, MO) and determination
of keratin was carried out.
Amino acid analysis
The dorsal body skin, foot scales and beak
of 16 days old embryos were hydrolysed
by 6M hydrochloric acid. of the selected
specimens were dissected and hydrolysed
by 6M hydrochloric acid. Sensitive amino
acids (especially Tryptophane and
cysteine) will be partially destroyed. The
samples were washed in hot dilute
detergent solution at neutral pH and rinsed
Materials and Methods
One hundred fertile eggs from in crossbred
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Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 315-327
in warm tap water and then distilled water.
Any pulpy protein in the coloumn was
squeezed out and extracted several times
with petroleum ether, followed by 95%
ethyle alcohol and allowed to dry in a
watch glass. The samples were dried
under vacuum, redissolved in 10 to 100 µl
0.2 M sodium citrate buffer, pH 2.0, and
loaded on the amino acid analyzer
equipped with a cation exchange column
(Amersham Pharmacia Biotech), which
was equilibrated in 0.2 M sodium citrate
buffer, pH 2.0. Elution was performed
with a gradient of pH and ionic strength as
instructed by the manufacturer. Detection
of the modified amino acids was achieved
calorimetrically at 440 nm for proline and
hydroxyproline and at 570 nm for all other
amino acids (Niece et al., 1991).
embryos were incised immediately at
developing stages and fixed in 10 %
formal saline and processed for
histological investigation and examined,
photographed under bright field light
microscopy.
Scanning
electron
investigation
microscopic
Dorsal body skin and foot scales of
developing chick embryos were separated
and immediately fixed in 2.5%
glutaraldhyde and 2% paraformaldhyde in
0.1 M cacodylate buffer (pH 7.4). After
washing the samples with deionized water,
they were stepwise dehydrated in 50%
(v/v), 80% (v/v), absolute ethanol. Drying
was done in a carbon dioxide critical point
drying apparatus. The samples were
mounted on aluminum stubs, coated with a
thin layer of gold by low voltage DC
sputtering and viewed using a Joel
5300JSM (Musashino 3-chome Akishima
Tokyo 196-8558, Japan).
SDS-PAGE analysis of keratin and
protein
Fresh biopsies of skin, foot scales and
beak at 9, 12 & 14-days old embryo were
examined by sodium dodecylsulfate
polymerase gel electrophoresis according
to the method of Laemmli (1970).
Electrophoresis was carried out at a
constant 200 V. The separated proteins
were placed on polyacrylamide gels
stained with Coomassie blue R-250 (60
mg/L) in an acidic medium for the
generation of an electrostatic attraction
between the dye molecules and the amino
groups of proteins (Andrews , "86). In
case of keratin electrophoresis, two kinds
with different molecular weight were used
as standard against the extracted protein
from the investigated specimens.
Transmission
investigation
electron
microscopic
Dorsal body skin and foot scales of
developing chick embryos were separated
and immediately fixed in 2.5%
glutaraldhyde and 2% paraformaldhyde in
0.1 M cacodylate buffer (pH 7.4). After
rinsing in 0.1 M cacodylate buffer, they
were post fixed in a buffered solution of
1% osmium tetra oxide at 4ºC for 1.5 hour
and dehydrated in ascending grades of
ethyl alcohol and embedded in epoxyresin. Ultrathin sections were cut with a
diamond knife on a LKB Ultratome IV
((LKB Instruments, Bromma, Sweden)
and mounted on grids, stained with uranyl
acetate and lead citrate, and examined
under a joel 100CX transmission electron
Light microscopic investigation
Dorsal body skin, foot scales and beak
region of 9, 12 & 14-days old chick
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microscope
(Musashino
3-chome
Akishima Tokyo 196-8558, Japan).
cone apex. The barbs appear intermingled
with each others (Figure. 3C). At 16 daysold, the feather proper structure is
detected. It is composed of a main shaft
from which emerged barbs and barbules.
The barbs and barbules are formed of
cylindrical articulated structures (Figures
3D-F).
Results and Discussion
Keratin and amino acids content
Figure (1,2) illustrated the keratin and
amino acids contents in dorsal feathery
skin, foot scales and beak respectively.
The keratin is markedly increased in foot
scales of 10-days old embryo comparing
with feathery skin and beak region. At 12
and 14-days old embryo, the keratin
attained highest increase in the beak
region. The amino acid contents of serine,
glutamine, prolline, glycine, alanine,
valine and leucine are detected at higher
concentration more than the other
estimated amino acids and varied
markedly between the mentioned regions.
The amino acids serine, glycine, alanine,
leucine
and isoleucine are markedly
increased in foot scales more than the
other body regions. There are least
variations of the others amino acid content
in feathery skin, foot scales and beak
regions.
At the light microscopic level, the feather
follicle starts in the form of a placode of
epidermal-dermal condensations at 9-days
old. Fibroblasts are more dense in the
dermis and infiltrated in between the
collagen fibrils and blood vessels (Figures
4A). The morphogenesis of follicles
proceeds in the subsequent stages 10 and
12-days old embryo. The feathers
elongated in proximal-distal axis and cross
sections possess regularly oriented barb
plates at different levels of the ramus
(Figures 4 B-D).
At transmission electron microscopic
level, 11-days old embryo possessed
thinned epidermis composed of two-cell
layers thick. The innermost cell layer is
stratum germinativum composed of more
active oval-shaped cells. Their cytoplasm
showed abundant rough endoplasmic
reticulum,
mitochondria
and
free
ribosomes. The epidermal-dermal junction
is irregular. The dermis is enclosed with
fibroblast cells. The other kinds of cells
are peripherally located and lacked the
presence of keratohyalin granules and
similar to stratum spinosum cells (Figures
6 A&B). At 14-days old embryo, the
epidermis is composed of four cell layers
thick. Both epidermal cells exhibited
abundant distribution of mitochondria,
rough endoplasmic reticulum and free
ribosomes in their cytoplasm. The
peripheral cell layer suspected to be
stratum granulosum cells as a result of
cytoplasmic distribution of
fine
keratohyaline granules (Figures 6 C,D ).
Scanning, light and transmission
electron microscopy of feathers
Scanning
electron
microscopic
observations of nine-day old embryo
revealed that, the feather buds primordia
forming apical ectodermal ridge as a result
of epidermal - dermal condensation.
(Figure. 3A). The feathers take cylindrical
pattern structure at 12 day-old chick
embryo (Figure. 3B).
At 14 days-old
chick embryo, the feather follicles possess
necrotic zones forming barbs at regular
spaces. The base of each feather filament
invaginated into the dermis forming
feather-epidermal collar at feather
epidermal junction. The terminal end of
the feather takes the criteria of feather
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Figure.1 Amino acids contents of feathers, beak and foot scales of 16 days chick embryo.
Ca.cy, cysteine; Asp, Asparagine; Thr, Threonine; Ser, Serine; Glu, Glutamine; Pro, Prolline;
Gly, Glycine; Ala, Alanine; Val, Valine; Iso.Le, Isoleucine; Leu, Leucine; Try,
tyrosine; Ph.ala, Phenylalanine; Arg., Arginine. Each result represents the mean ±
standard error of five replicates.
Figure.2 SDS-PAGE of keratin analysis in dorsal skin of foot scale of
developing chick embryos.
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Figure.3 (A-F) Electron micrographs of developing feathers of chick embryos.
A.Nine-days old. B. Twelve-days old. C. Fourteen-days old. D. E & F. Sixteen-days old.
(Abbreviations: B, Barb, CAB, Cylindrical articulated barb; MA, Main axon; FF, Feather
filament; FP, Feather papilla; OV, Opened vans ).
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days-old embryo revealed that the
epidermis is composed of several cell
layers thick. The stratum germinativum
possessed characteristic hemidesosomes
infiltrated their parietal surfaces. The
stratum spinosum cell oval-shaped and
has centrally located nuclei with two or
three nucleoli. The intercellular space is
enclosed by threads of keratohyaline
granules.
Scanning, light and transmission
electron microscopy of foot scales
Scanning electron microscopy exhibited
SEM level, more organized foot plate in
the nine-days old embryo. The epidermis
formed the placaode and interplacode cell
populations. The primordial structures of
the scutate scales are detected (Figures 5A,
B).
At the 12-days old embryo,
morphogenesis of scutate scales appeared
in the form of folded ridges over the toes
surface. The scutate scales are large and
rectangular in shape and overlapping with
each other. Cornification of the claw is
proceeded (Figures 5C-F). At the 14-days
old embryo, the dorsal foot surface is
composed of overlapping scutate scales.
The scutella scales are distributed lateral
to the scutate scales and are smaller in
size. The third types, reticulate scales are
detected on the foot pad and characterized
by their radial symmety (Figures 5 G-I ) .
At the 16-days old embryo, the different
parts of the tarsal shank are covered with
several sheets of cornification (Figures 5 J,
K ).
Toward the peripheral surface, the stratum
granulosum is more recognized with
peculiar large keratinocytes having
indented nuclear envelope rich of
euchromatin and containing one nucleoli.
Their cytoplasm as well as the intercellular
space is rich in keratohyaline granules of
both vesicular characteristic structure and
varying sizes. Cornification of the outer
surface is more detected. The keratinized
layers are composed of several layers and
enclosed by newly formed and dead
corneocytes. The epidermal dermal
junction is markedly folded. The dermis
possesses abundant distribution of
collagenous fibrils, fibroblasts and blood
vessel (Figures 6 A1-D1).
At the Light microscopic level, 12-days
old chick embryo, foot region exhibited
different pattern structure of epidermal
scales. Scutate scales are the largest form
and have both similar thickness of their
apex and hinge region formed of two-cell
layers thick. Scutella represents the second
types of scales and is detected in the
ventral surface. The scales appeared more
flatter and regular oriented but lack
overlapping pattern structure as scutate
scales. A third type is reticulate scale in
the lateral surface. (Figures 4 A1,B1 ). At
14 and 16-days old embryo, keratinization
of the epidermis of the different types of
scales is detected. (Figures 4 C1-E1 ).
During development of chick embryos, the
integument possessed diversity of
epidermal structures in abdomen, foot and
beak region. Although they are markedly
different,
they
share
common
developmental pathways. The observed
findings of skin development of both
abdomen and foot region revealed that
they showed the regular sequence of
growth pattern, including the formation of
a placode stage involving sequences of
invagination in the dermis at nine days.
However, with the subsequent developing
ages, the growth patterns are markedly
deviated. The feather germs grow in a
proximal-distal axis forming a tube with
Ultrastructural observations of scales of 14
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Figure.4(A-C3) Histological micrographs of feather (A-A2), foot scales (B-B3) and beak of
chick embryo.
A. Feather histogenesis of 9-days old. A-A2. Feather histogenesis of 10 days-old. B. Foot
scale histogenesis of 9 days old embryo B1-B3. Foot scale histogenesis of 16 days old
embryo. C-C1.Beak cornification of 9 days old embryo. C2C3. Beak cornification of 10 days
old embryo (Abbreviations: B, Barbs; C, Cornification; D, Dermis; R, Reticulate scale; SC,
stratum corneum; SS, Scutate scale; Sc, Scutella ).
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Figure.5(A-I) Electron micrographs of foot scales of developing chick embryo.
A&B. Nine-days old. C- E. Twelve-days old embryo. F-H. Fourteen-days old. I. Sixteendays old.( Abbreviations: Cl, Claw; R, Reticulate scale; SS, Scutate scale; Sc, Scutella; SKs,
Shedding keratin).
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Figure.6 (A-D& A1-D1) Electron micrographs of dorsal body skin (A-D) and foot scales
(A1- E1). A& B.Epidermal cell layers of 11 days old embryo. C & D. epidermal cell layer of
12 days old embryo. A1-D1.Epidermal cell layers of foot scales of 14 days old embryo.
Note: Keratinocytes and cytoplasmic inclusions of keratohyaline granules. (Abbreviations:
KF, keratin Filament; KG, Keratohyaline granules; M, Mitochondria; N, Nucleus; RER,
rough endoplasmic reticulum; SG, Stratum germinativum; SGr, Stratum granulosum; SS,
Stratum spinosum).
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early origin of barbs followed by regularly
oriented parietal surface of the follicle
tube at 10 & 12-days old. The epidermis is
differentiated into two or three cell layers
thick having the innermost germinativum
cells, and the outermost is the perspective
keratinocytes. Cornification is less
detected in the epidermis of skin
comparing with that of the feather. Also,
the epidermal placode of foot is further
developed into three kinds of scales
scutate, scutella and reticulate scales. The
beak
shows
another
pattern
of
developmental programmes, employing
epithelial and mesenchymal cells to
produce composite structures, forming the
architecture structure specific of chick
with a glance cornification of its outer
covering sheath.The present findings agree
with the findings of Dhouailly (1978), Wu
et al., (2004), Schneider (2005) and
Widelitz et al., (2006).
main shaft from which emerged barbs and
barbules. The barbs and barbules appeared
in the form of cylindrical articulated
structures. The structural form of avian
scales comprised three main types, scutate,
scutella and reticulates. They composed
mainly of almost similar thickness of their
outer scale surface and hinge region
reflected absence of articulation of the
scales as in reptilian section. The
epidermis is composed of several cell
layers thick with the marked degree of
cornification being at the highest grade in
the beak region. The dermis is denser of
fibroblast and collagen fibrils.
The present findings supported the work
of O Guin et al., (1982), Haake et al.,
(1984) and Haake, (1985). According to
Fisher et al., (1988), Chuong (1998) and
Chuong et al. (2003), despite diversity of
epidermal appendages, they have many
similar developmental origins.
According to Webb and Noden (1993) and
Schneider (2005), the variations of beak
structure from feathers started from earlier
embryonic development and formation of
primordial tissue. The upper beak region is
derived from the frontonasal and paired
maxillary primordia, whereas the lower
portion forms from paired mandibular
primordia. There was a patterning
potential of cranial neural crest
mesenchyme during beak morphogenesis
(Schneider and Helms, 2003).
SEM observations of feather and scale
development
during
embryonic
development
reflected
the
closely
homologous of their early cone
differentiation pattern; however, diversity
occurred of their initial formation.
Feathers primordia showed a similar
characteristic
structure
in
light
microscopic level, however, striking
findings at the 14-days old, the feather
proper tubular structure is composed of the
In addition, we observed marked
variations of epidermal thickness between
abdominal skin feathers, foot skin and
beak region, being more thickened in beak
and foot skin. The peripheral cell layers of
foot regions characterized by increase of
keratinocytes and keratohyaline granules
of different size. The beak epidermis
becomes
more
cornfield
with
characteristic glossy appearance acquiring
the structural appearance of the beak
required for feeding habit.
Also, we detected that the biochemical and
molecular analysis of keratin, amino acids
and
protein
expression
confirmed
variations of keratinization of the
epidermal appendages. Foot scales
exhibited highest keratin formation at 10day old embryos in comparison with
abdominal skin and beak regions.
However, keratin content attained more
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Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 315-327
increase in the beak region at 12 and 14day old embryo. The amino acid contents
of serine, glutamine, prolline, glycine,
alanine, valine and leucine are detected at
higher concentration in foot scales more
than that of other body regions. Although
there is a relative similar arrangement of
amino acids, their concentration varied
according to the degree of cornification in
different epidermal regions, which is
parallel
with
the
function
of
developmental part either in feeding for
beak or protection from damage as in the
foot region.
the sky: Defining the feather with
integument fossils from mesozoic
China and experimental evidence from
molecular laboratories. J. Exp. Zool.
Part. B. Mol. Develop. Evol. 298: 4256.
Chuong, C.M., and Widelitz, R.B. 1998
Feather morphogenesis: a model of the
formation of epithelial appendages, in
Molecular
Basis
of
Epithelial
Appendage Morphogenesis (Chuong,
C.M., ed.), Landes Bioscience, Austin,
T.X.
Dalla Valle, L., A. Nardi, V. Toffolo, C.
Niero, M. Toni and Alibardi, L. 2007.
Cloning and characterization of scale
beta-keratins in the differentiating
epidermis of geckoes show they are
glycine-proline-serine-rich
proteins
with a central motif homologous to
avian
beta-keratins.
Develop.
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Dalla Valle, L., V. Toffolo, V. Belvedere
and Alibardi, L. 2005. Isolation of a
mRNA encoding a glycine-proline-rich
beta-keratin
expressed
in
the
regenerating epidermis of lizard.
Develop. Dynamic. 234:934 947.
Dhouailly, D., 1978. Feather-forming
capacities of the avian extraembryonic somatopleure. J. Embryol.
Exp. Morphol. 43: 279-87.
Dreher, F., A. Arens, J.J. Hostýnek, S.
Mudumba, J. Ademola and Maibach,
H.I. 1998. Colorimetric method for
quantifying human Stratum corneum
removed by adhesive-tape stripping.
Acta. Dermatol. Venereol. 78:186-189.
Fisher, C.J., L.W. Knapp and Sawyer,
R.H. 1988. Retinoic acid induction of
featherlike structures from reticulate
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Fraser, R.D., and Parry, D.A. 1996. The
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I. J. Biol. Macromolecul.19: 207-211.
Gregg, K., and Rogers, G.E. 1986. Feather
According to recent molecular biology and
proteomic studies carried out by Dalla
Valle et al., (2005, 2007), the highest
concentrations of serine, glutamine,
prolline, glycine, alanine, valine and
leucine reflected the degree of keratin
formation. The homology of the core-box
among different epidermal appendages
species suggested that this amino acid
sequence and concentrations evolve from a
progenitor sequence present in the stem of
avian skin. The core-box of amino acids is
implicated in the formation of keratin
filaments of scales, claws, and feathers
(Gregg and Rogers, 1986; Fraser and
Parry, 1996).
Finally, the authors concluded that, the
integument
structures
have
close
similarities in their early origin but
markedly different in their initial
destination in both fine structures and their
keratin and amino acids contents.
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